title: Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin.
abstract: MarR is the dedicated autorepressor of the marRAB operon found in seven genera of the Enterobacteraceae. The MarA transcriptional regulator directly activates numerous genes involved in multidrug resistance and other environmental responses. MarR is inactivated by certain phenolic ligands, such as salicylate, by an unknown mechanism. Our recent work has shown that several amino acid residues of Escherichia coli MarR affecting ligand binding are located between the dimerization and DNA-binding domains. To further characterize the ligand-binding region of MarR, we have now examined 7 point mutants generated by random mutagenesis and 11 site-directed alanine replacement mutants for inactivation by three ligands: salicylate, 2,4-dinitrophenol, and plumbagin. Inactivation of MarR was quantitated in intact cells by loss of MarR-mediated repression of a chromosomal mar-lacZ transcriptional fusion. The results showed that most of the residues important for ligand effectiveness lay in the α1 and α2 helices of MarR, between the putative DNA-binding domain and the dimerization domain of MarR, reinforcing our earlier findings. Moreover, the three ligands had different, but overlapping, sets of residues impacting their effects on MarR.
authors.affiliation: Department of Molecular Biology and Microbiology, Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, Boston, MA, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: McMurry
authors.forename: Stuart B
authors.initials: SB
authors.lastname: Levy
mesh_terms: D019297:2,4-Dinitrophenol / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009285:Naphthoquinones / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012459:Salicylates / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D008024:Ligands; C080004:MarR protein, E coli; D009285:Naphthoquinones; D012097:Repressor Proteins; D012459:Salicylates; C093326:mprA protein, E coli; D019297:2,4-Dinitrophenol; C014758:plumbagin
keywords: error-prone PCR; induction; ligand; marRAB operon; mutagenesis; repression